By employing a microdissection technique and sensitive radiometric assays, the distributions of acetylcholine (ACh), choline acetyltransferase (CAT), and acetylcholinesterase (AChE) were examined in 60 discrete areas of rat forebrain. A 5-fold range was observed for ACh levels, while that for the cholinergic enzymes was about 100-fold. AChE activity was always higher than that of CAT. ACh levels did not appear to relate to the activity of either enzyme in a simple manner. However, there was a good overall correlation between CAT and AChE activities. Both ACh and the enzymes were concentrated in the striatum. The highest forebrain CAT activity was found in the n. habenulae medialis. One or more of the cholinergic parameters occurred in moderate to high amounts in several limbic structures. These included the n. accumbens, olfactory tubercle, septum, n. tractus diagonalis, piriform and entorhinal cortices, and some thalamic and amygdaloid nuclei. In the hypothalamus, substantial CAT activity was found in the median eminence. Such biochemical measurements should prove valuable in future experiments designed to determine cholinergic function and localize cholinergic pathways. BIBLIOGRAPHIC REFERENCES: Jacobowitz, D.M. and Goldberg, A.M.: Determination of acetylcholine in discrete regions of the rat brain. Brain Res. 122: 575-577, 1977. Hoover, D.B., Muth, E.A., and Jacobowitz, D.M.: A method for sectioning microwave-fixed brain prior to microdissection and acetylcholine analysis. Neuroscience Letters, 1977 (in press).